Method for separating cell from biological tissue

ABSTRACT

Provided is a method for efficiently and stably separating cells having a high biological activity from a biological tissue, by using a degrading-enzyme composition, which is prepared by adding an enzyme for degrading a major protein of the biological tissue in an amount determined depending on the composition of the major protein to a predetermined amount of a neutral protease and/or a protease derived from  Clostridium  sp. According to this method, the type and amount of protein-degrading enzyme to be used for isolating cells can be determined from the composition of a major protein of the biological tissue. Thus, cells having a high biological activity can be efficiently separated while reducing the amount of protein-degrading enzyme to be used.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/109,242, filed Aug. 22, 2018, which is a continuation of U.S.application Ser. No. 14/765,020, filed Jul. 31, 2015, which is anational stage application of International Appl. PCT/JP2014/000527,filed Jan. 31, 2014, and which claims priority of Japanese Patent Appl.No. 2013-018774, filed Feb. 1, 2013, all of which are herebyincorporated by reference.

TECHNICAL FIELD

The present invention relates to a method for separating cells from abiological tissue, and particularly to a method for efficiently andstably separating cells having a high biological activity by controllingthe amount of protein-degrading enzyme to be used.

BACKGROUND ART

Enzymatic isolation of cells or cell aggregates (cell clusters), whichare present in various organs and tissues such as liver, pancreas,kidney, periodontal tissue, skin, cartilage, bone and nervous tissue ofmulticellular animals such as mammals, birds, reptiles and fish as wellas in an ES cell tissue and a fibroblast cell tissue serving as amaterial for inducible pluripotent stem cells (iPS cells), is useful ina wide variety of usages including cell transplantation, cell strainestablishment, therapy, diagnosis, and examination. For example,diabetes is treated by a medical procedure in which pancreatic isletsare isolated from a pancreas and transplanted to a patient. Forpancreatic islet transplantation, it is essential to separate a cellaggregate called as pancreatic islets present in the pancreas tissue. Atthis time, it is necessary to separate pancreatic islets by degradingthe pancreas tissue without damaging the pancreatic islets. Furthermore,liver failure and the like are treated by separating liver cells from aliver and transplanting them into a patient.

In order to dissociate a biological tissue and separate cells or cellaggregates contained in the biological tissue, it is necessary toseparate cells or the like to a desired level and separate the cellsfrom the other portion of the biological tissue. In separating cells orthe like from a biological tissue by enzymatic isolation, it isnecessary to treat intercellular matrix with a protein-degrading enzymesuch as collagenase.

A biological tissue is constituted of cells and intercellular matrix(intercellular substance). The intercellular substance is a substanceanchoring cells and includes a structural material and a non-structuralmaterial. Examples of the structural material include fibers such as acollagen fiber, an elastic fiber and a fine retinal fiber. Examples ofthe non-structural material, which is a component filling the spacebetween fibers, called as a substrate and refers to sol or gelsubstance, include a sugar protein and a proteoglycan.

A typical intercellular substance is a protein called collagen whichrepresents about ⅓ of in-vivo total protein weight. Collagen has a fiberstructure and is officially called as a collagen fiber or a collagenousfiber.

The tissues are roughly classified into four categories: an epithelialtissue, a supporting tissue, a muscular tissue and a nervous tissue. Theepithelial tissue is a tissue covering the surface of a body or organsand consisting of highly dense cells without the intercellular substanceinterposed therebetween. The supporting tissue, which plays a role insupporting e.g., a body, organs and cells, includes a connective tissue,a cartilaginous tissue, a bone tissue, blood and lymph. The musculartissue is integrally formed of cells differentiated for the purpose ofcontraction motion, in which the ratio occupied by the intercellularsubstance is extremely low. The muscular tissue is constituted of musclecells, a connective tissue, blood vessels and nerves; however, a majorstructure thereof is a muscle fiber. The nervous tissue is primarilyconstituted of the endoneurium and the perineurium, each containing alarge amount of intercellular substance (collagen).

The connective tissue, which is a kind of supporting tissue, refers toan adipose tissue and a fibrous connective tissue. As the structuralcomponents of the fibrous connective tissue, a collagen fiber and anelastic fiber are mentioned. The fibrous connective tissues are roughlydivided into a hydrophobic connective tissue and a dense connectivetissue. The hydrophobic connective tissue refers to a fibrous connectivetissue having collagen fibers irregularly arranged therein anddistributed in e.g., a subcutaneous tissue, a mucous membrane tissue,nerve, adventitia, and an interlobular tissue.

A peptide chain of collagen characteristically have a primary structurein which glycine repeatedly appears every third residue, like“-glycine-amino acid X-amino acid Y”. It is known that there are about30 types of molecule species in human collagen. The collagen mostabundantly present in a body is fibrous I-type collagen. Non-fibrouscollagen IV is also abundantly contained. Collagen IV fibers are linkedwith each other via an intermolecular disulfide bond and participated information of a reticular tissue (Non Patent Literature 1). It has beenreported that collagen VI is present between the pancreatic islets andthe endocrine tissue (Non Patent Literatures 2 and 3).

As to the enzymes for degrading tissue, various types of crudecollagenases derived from Clostridium histolyticum contain not onlycollagenase but also various proteases (having collagen degradationactivity and nonspecific protein degradation activity) and non-proteasecomponents (e.g., phospholipase). By virtue of the crude collagenase,cells and cell populations are enzymatically separated from almost allbiological tissues.

It is known that a pancreatic islet tissue is also degraded by aprotein-degrading enzyme selected from the group consisting of two typesof collagenases (Col G and Col H) derived from Clostridium histolyticum,a protease (thermolysin, Dispase or neutral protease (NP) derived fromClostridium histolyticum) (Patent Literatures 1 and 2). For example, twotypes of collagenases and a neutral metallo protease produced byClostridium histolyticum are known to be preferably used (Non PatentLiterature 4). In enzymatically separating cells or the like from abiological tissue, two types of collagenases are reported to haveimportant roles in attaining the yield and keeping biological activityof the cells or the like to be separated (Non Patent Literature 5).

It is also reported that the number of pancreatic islets separated froma human pancreas increases in proportion to an α-N-benzoyl-L-arginineethyl ester hydrochloride (BAEE) degradation activity that an enzymemixture exhibits (Patent Literature 1 and Non Patent Literature 6). Asthe enzyme having a BAEE degradation activity, clostripain (CP) producedby Clostridium histolyticum is known.

The state of intercellular matrix in a biological tissue (particularly,collagen content) varies depending upon the organism species, age, sex,tissue, living environment and etc. How much collagen is contained inwhich state of matrix of which tissue has not yet been elucidated.Collagen is present in a wide variety of species from mammals to fishand difference in constituent collagen between species has not yet beenelucidated.

It seems to be possible that whether or not a specific type of collagenis present in the matrix of a biological tissue is determined byimmuno-staining using antibodies against individual types of collagens.However, since many types of collagens are present and collagens arepresent in a wide variety of multicellular animals, it is difficult toproduce antibodies. Because of these problems, it is difficult torealize identification of the specific type of collagen.

CITATION LIST Patent Literature

Patent Literature 1: International Publication No. 1996/00283

Patent Literature 2: International Publication No. 1998/24889

Non Patent Literature

Non Patent Literature 1: Inoue et al., J Cell Biol, 97, 1524-1539 (1983)

Non Patent Literature 2: SJ Hughes, P McShane, Transplant Proceedings,37, 3444-34445 (2005)

Non Patent Literature 3: SJ Hughes, A Clark, P McShane, Transplantation,81(3) 423-426 (2006)

Non Patent Literature 4: E Linetsky et al., Diabetes, 46, 1120-1123(1997)

Non Patent Literature 5: D Brandhorst et al., TransplantationProceedings, 37(8), 3450-3451 (2005)

Non Patent Literature 6: H Brandhorst et al., Transplantation, 87(3),370-375 (2009)

SUMMARY OF INVENTION Technical Problem

In separating cells or cell aggregates from a biological tissue, it isnot easy to degrade only various structural proteins constitutingintercellular matrix of the biological tissue and organs with aprotein-degrading enzyme without degrading and damaging the surface oftarget cells or the like to be separated. As described above, the state(particularly, collagen content) of intercellular matrix in a biologicaltissue varies depending upon the organism species, age, sex, tissue,living environment, etc. Particularly, collagen significantly changes inphysical properties depending upon aging.

It is known that a human organ acts differently in response to aprotein-degrading enzyme depending upon e.g., the age, sex, habit andmedical history; however, a means for determining the type and amount ofoptimal protein-degrading enzyme has not so far been developed. In thepresent situation, isolation had to be performed by empiricallydetermining the type of enzyme and reaction time thereof. Pancreaticislets are separated in accordance with a protocol, more specifically, amedical worker treats a pancreas with a predetermined amount of enzymeonly by varying the time of degradation while visually checking thedegree of degradation of the pancreas. For this reason, the quantity andquality of the pancreatic islets to be obtained inevitably variesdepending upon the state of a target pancreas.

In view of the above circumstance, a main object of the presentinvention is to provide a cell separation method by which cells having ahigh biological activity can be efficiently and stably separated from abiological tissue.

Solution to Problem

In order to solve the aforementioned problems, the present inventionprovides the following methods.

[1 ] A method for separating cells from a biological tissue, using adegrading-enzyme composition, which is prepared by adding an enzyme fordegrading a major protein of the biological tissue in an amountdetermined depending on the composition of the major protein to apredetermined amount of a neutral protease and/or a protease derivedfrom Clostridium sp.

[2 ] The method according to [1], including a step of determining thecomposition of the major protein of the biological tissue; a step ofpreparing a degrading-enzyme composition by adding an enzyme fordegrading the major protein in an amount determined depending on thecomposition determined to the predetermined amount of a neutral proteaseand/or a protease derived from Clostridium sp.; and a step of treatingthe biological tissue with the degrading-enzyme composition prepared.

According to this method, the type and amount of protein-degradingenzyme to be used for isolating cells can be determined based on thecomposition of the major protein of a biological tissue. Thus, cellshaving a high biological activity can be efficiently separated bycontrolling the amount of protein-degrading enzyme to be used.

[3] The method according to [1] or [2], in which the enzyme fordegrading the major protein is collagenase H and/or collagenase G ofClostridium sp.

[4] The method according to any of [1] to [3], in which the majorprotein is collagen I and/or collagen III.

[5] The method according to [4], in which the amount ratio ofcollagenase H and/or collagenase G to be added to the degrading-enzymecomposition is determined in accordance with the composition of collagenI and/or collagen III.

[6] The method according to any of [1] to [5], in which the neutralprotease is thermolysin or a neutral protease derived from Clostridiumsp.

[7] The method according to any of [1] to [6], in which the proteasederived from Clostridium sp. is a protease having anα-N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) degradationactivity.

[8] The method according to any of [1] to [7], in which the proteincomposition of the biological tissue is determined by an enzymaticimmunoassay.

[9] A method for separating pancreatic islets from a pancreas tissue,using a degrading-enzyme composition, which is prepared by addingcollagenase H and/or collagenase G derived from Clostridium sp. in anamount sufficient to degrade collagen I and/or collagen III and inaccordance with a composition of collagen I and/or collagen III of thepancreas tissue, to a predetermined amount of a neutral protease and/ora protease derived from Clostridium sp.

[10] The method according to [9], in which the weight ratio (H/G) ofcollagenase H and collagenase G in the degrading-enzyme composition is0.35 or more.

[11] The method according to [9] or [10], in which the degrading-enzymecomposition contains a neutral protease derived from Clostridium sp. asthe neutral protease, and a protease having an α-N-benzoyl-L-arginineethyl ester hydrochloride (BAEE) degradation activity as the proteasederived from the Clostridium sp.

[12] A method for separating liver cells from a liver tissue, using adegrading-enzyme composition, which is prepared by adding collagenase Hand/or collagenase G of Clostridium sp. in an amount sufficient todegrade collagen I and/or collagen III and in accordance with acomposition of collagen I and/or collagen III of the liver tissue, to apredetermined amount of a neutral protease and/or a protease derivedfrom Clostridium sp.

[13] The method according to [11], in which the weight ratio (H/G) ofcollagenase H and collagenase G in the degrading-enzyme composition is0.25 or less.

[14] A method for separating pancreatic islets from a pancreas tissue,using a degrading-enzyme composition containing a neutral proteasederived from Clostridium sp. and a protease having anα-N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) degradationactivity.

[15] The method according to [14], in which the protease having the BAEEdegradation activity is clostripain.

In the present invention, the “biological tissue” includes, but notparticularly limited to, various organs and tissues of multicellularanimals such as mammals, birds, reptiles and fish. Examples thereof mayinclude liver, pancreas, kidney, periodontal tissue, skin, cartilage,bone and nervous tissue. Furthermore, an ES cell tissue and a fibroblastcell tissue serving as a material for an inducible pluripotent stemcells (iPS cells) are included in the “biological tissue”.

The “cells” include cells and cell aggregates and all types of cellspresent in the above biological tissues may be included. Examples of the“cells” may include hepatocytes, pancreatic islets and glomeruli.

Advantageous Effects of Invention

Owing to the present invention, a method for efficiently and stablyseparating cells having a high biological activity from a biologicaltissue is provided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the yield of pancreatic islets (ReferenceExample 1).

FIG. 2 is a graph showing measurement results of the ADP/ATP value ofthe pancreatic islets separated (Reference Example 1).

FIG. 3 is a graph showing measurement results of the ATP/DNA value ofthe pancreatic islets separated (Reference Example 1).

FIG. 4 is a graph showing measurement results of the Insulin/DNA valueof the pancreatic islets separated (Reference Example 1).

FIG. 5 is a graph showing evaluation results of the stimulation index ofthe separated pancreatic islets in SGS test (Reference Example 1).

FIG. 6 is a graph showing measurement results of the amount of TMPPattached to collagen I and collagen III degraded by collagenase H(Reference Example 2).

DESCRIPTION OF EMBODIMENTS

Now, a preferred embodiment for carrying out the present invention willbe described below. Note that the embodiment described below is atypical embodiment of the present invention and should not be construedas limiting the scope of the present invention.

The present inventors found that, of various collagen degrading enzymes,collagenase H (ColH) is a very important enzyme in degrading abiological tissue (see Reference Example 1 described later). Since ithas been considered in the art that collagenase G (ColG) is moreimportant in degrading a biological tissue (see Patent Literatures andNon Patent Literatures in the above), the finding is a matter ofsurprise. The present inventors have also found that Col H degradescollagen I and collagen III, which are major proteins in a biologicaltissue (see Reference Example 2 described later).

The present inventors have also revealed that if the abundance ofcollagen I and collagen III, which are major proteins that Col Hdegrades, in a biological tissue is measured, the amount ofprotein-degrading enzyme suitably used for separating cells from thebiological tissue can be previously determined, with the results thatthe cells can be separated without excessively being damaged (seeExample 1). The present inventors have further found that a neutralprotease (NP) of Clostridium sp. serving as a neutral protease and aprotease having a BAEE degradation activity synergistically work toenhance the efficiency of separating pancreatic islets from a pancreastissue (see Example 2).

In this way, a method for separating cells from a biological tissueusing a degrading-enzyme composition, which is prepared by adding amajor protein-degrading enzyme of the biological tissue in an amountdetermined depending on the composition of the major protein, wasconceived. According to the method for separating cells, it is possibleto determine the type and amount of protein-degrading enzyme to be usedfor isolating cells based on the composition of the major protein of thebiological tissue. Thus, cells having a high biological activity can beefficiently separated while reducing the amount of protein-degradingenzyme to be used.

Major Protein

In the method for separating cells according to the present invention,the major protein is a protein constituting intercellular matrix and maypossibly vary depending upon the target biological tissue. Examples ofcollagen include collagen I, collagen II, collagen III, collagen IV,collagen V, laminin, fibronectin and vitronectin. For example, whencells are separated from a pancreas tissue and a liver tissue, the majorprotein preferably includes collagen I and collagen III.

Composition of Major Protein

In the method for separating cells according to the present invention,the composition of the major protein refers to the ratio of the majorprotein in the proteins constituting intercellular matrix or the amountthereof. For example, when cells are separated from a pancreas tissueand a liver tissue, the composition of major protein refers to theratios or amounts of collagen I, collagen II, collagen III, collagen IV,collagen V, collagen VI, laminin, fibronectin and vitronectin; andparticularly refers to the ratios or amounts of collagen I and collagenIII.

The composition of the major protein can be determined by enzymaticimmunoassay using a specific antibody against the major protein and aspecific inhibitory peptide thereof. More specifically, a labeledantibody or inhibitory peptide is allowed to react with a biologicaltissue slice, for example, by an immunohistological approach. Next, theintensity of a signal from the label attached to the antibody orinhibitory peptide bound to the major protein is visually checked underobservation of a microscope or detected by an apparatus. In this manner,the major protein is quantified and the composition is determined. Thecomposition of the major protein can be also determined quickly by e.g.,a sedimentation reaction in gel, immunoelectrophoresis, animmunoturbidimetric method or an enzyme immunoassay using an antibody.

Major Protein-Degrading Enzyme

In the method for separating cells according to the present invention, amajor protein-degrading enzyme may possibly vary depending upon thetarget biological tissue. For example, when cells are separated from apancreas tissue and a liver tissue, the degrading enzymes for a majorprotein are Col H and Col G. When pancreatic islets are separated from apancreatic islet tissue, Col H is important as a major protein-degradingenzyme; whereas when liver cells are separated from a liver tissue, ColG is important as a major protein-degrading enzyme.

Degrading-Enzyme Composition

The degrading-enzyme composition to be used is preferably prepared byadding the aforementioned major protein-degrading enzyme to apredetermined amount of a neutral protease and/or a protease derivedfrom Clostridium sp.

As the neutral protease, for example, thermolysin or a neutral protease(NP) derived from Clostridium sp. is mentioned.

As the protease derived from Clostridium sp., a protease having anα-N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) degradationactivity is mentioned. The protease having a BAEE degradation activityis preferably clostripain (CP). Since Clostridium sp. strain producesvarious types of proteases, an enzyme produced as a recombinant proteinis desirably used as the protease derived from Clostridium sp. strain.

The degrading-enzyme composition to be used for separating pancreaticislets from a pancreas tissue preferably contains both a neutralprotease (NP) derived from Clostridium sp. as a neutral protease and aprotease having a BAEE degradation activity as the protease derived fromClostridium sp. NP and the protease having a BAEE degradation activitysynergistically work to exert an effect of increasing the number ofpancreatic islets to be separated from a pancreas tissue.

The amount of a neutral protease and/or a protease derived fromClostridium sp. in a degrading-enzyme composition, which is notparticularly limited, is, for example, 0.3 to 0.5 mg per composition (10mL). Note that the content of the enzyme in a degrading-enzymecomposition may appropriately be controlled also in accordance with theweight of the target biological tissue to be treated.

The amount of major protein-degrading enzyme added to a degrading-enzymecomposition is determined based on the composition of the aforementionedmajor protein. More specifically, the amount of major protein-degradingenzyme is determined based on the ratio or amount of major protein inthe proteins constituting intercellular matrix. More specifically, forexample, if the proteins constituting the intercellular matrix areCollagen III and collagen I, the amounts of Col H and Col H to be usedare determined based on the ratios or amounts of collagen III andcollagen I. Particularly favorably, the amount ratios of Col H and Col Gto be used in the degrading-enzyme composition are determined based onthe ratios or amounts of collagen III and collagen I.

For example, when pancreatic islets are separated from a pancreas tissuein which the ratios of collagen III and collagen I in the proteinsconstituting intercellular matrix are high (amounts are large), theamount of Col H to be used is preferably 0.35 times or more as large asthat of collagenase G. Note that pancreatic islets can be separated byusing a degrading-enzyme composition containing only collagenase H (ofcollagenase G and collagenase H) although separation efficiencydecreases. In contrast, when liver cells are separated from a livertissue in which the contents of collagen III and collagen I are small,the amount of Col H to be used is preferably 0.25 times or less as smallas that of collagenase G.

For example, when pancreatic islets are separated from a pancreastissue, a degrading-enzyme composition can be prepared by adding, tothermolysin (0.3 mg), high purity Col G (2 mg) and

Col H (1.1 mg) (0.55 times as large as collagenase G) produced byrecombination per composition (10 mL). Furthermore, when liver cells areseparated from a liver tissue, a degrading-enzyme composition can beprepared by adding, to thermolysin (0.5 mg), high purity Col G (14.4 mg)and Col H (3.6 mg) (0.25 times as large as collagenase G) percomposition (10 mL).

Enzyme Treatment

A biological tissue may be treated with a degrading-enzyme compositionin the same manner as in a conventional method. The degrading-enzymecomposition contains a major protein-degrading enzyme in an amount (theminimum requisite amount) required for degradation of the biologicaltissue based on the composition of the major protein of the biologicaltissue. Thus, desired cells can be separated from a biological tissuewithout producing a problem such as a reduction of physiologicalactivity of separated cells and a reduction of yield caused by anexcessive or insufficient content of enzyme in a degrading-enzymecomposition.

As described above, according to the method for separating cellsaccording to the present invention, cells having a high biologicalactivity can be efficiently and stably separated from a biologicaltissue by determining the composition of the major protein in abiological tissue and preparing a degrading-enzyme composition by addingthe enzyme for degrading the major protein in a requisite amount. Inshort, according to this method, high quality cells can be separated ina high yield by applying an optimal enzymatic treatment to any targetbiological tissue.

EXAMPLES Reference Example 1: Identification of Major Protein-DegradingEnzyme to be Used in Separating Cells or the Like from Biological Tissue

[Identification of Enzyme for Use in Separating Pancreatic Islets fromRat's Pancreas Tissue]

A male Lewis rat (10 to 13 weeks old) was used. Before the pancreas wasexcised out, the duodenum was clamped. A degrading-enzyme composition(10 mL, cold Hank's Balanced Salt Solution (HBSS)), which contains atleast one of a protein-degrading enzyme: recombinant collagenase G (8.4mg) and collagenase H (2.9 mg), and a neutral protease: thermolysin (0.3mg), was injected through the bile duct. After a treatment was performedat 37° C. for 14 minutes, density gradient centrifugation was performedto obtain a fraction of pancreatic islets.

The case (GH group) where thermolysin, collagenase G and collagenase Hwere simultaneously added, the case (G group) where thermolysin andcollagenase G alone were added, and the case (H group) where thermolysinand collagenase H alone were added, were evaluated for yield and abiological activity of pancreatic islets. The results are shown in FIGS.1 to 5. In the SGS test (FIG. 5), the volume ratio of insulin secretedat the time of exposure to high concentration glucose (16.7 mM) andinsulin secreted at the time of exposure to low concentration glucose(1.67 mM) was obtained and used as a stimulation index for evaluation.

As shown in FIG. 1, pancreatic islet yield (islet equivalents (IEQs)) ofthe H group was 70% of that of the GH group. In contrast, in the Ggroup, none of pancreatic islets were separated. As is apparent from theADP/ATP value of pancreatic islets (FIG. 2), ATP/DNA value thereof (FIG.3), insulin/DNA value thereof (FIG. 4) and stimulation index (FIG. 5) inthe SGS test, the pancreatic islets obtained in the H group had the samephysiological activity as those of the pancreatic islets obtained in theGH group. From these results, collagenase H was identified as the majorprotein-degrading enzyme to be used in separating pancreatic islets froma pancreas tissue.

[Identification of enzyme for use in separating liver cells from rat'sliver tissue]

A degrading-enzyme composition (10 mL, HBSS), which contains at leastone of a protein-degrading enzyme: recombinant collagenase G (14.4 mg),collagenase H (3.6 mg), and a neutral protease: thermolysin (0.5 mg),was prepared. The liver tissue (10 to 12 g) was treated with thedegrading-enzyme composition prepared at 37° C. for 7 minutes and thensubjected to density gradient centrifugation to separate liver cells.

In the case (GH group) where a degrading-enzyme composition prepared byblending collagenase G and collagenase H with thermolysin was used,5.02±2.11×10⁸ of liver cells were successfully separated. In the case (Ggroup) where a degrading-enzyme composition prepared by addingcollagenase G to thermolysin was used, 0.81±0.11×10⁶ of liver cells weresuccessfully separated. In contrast, in the case (H group) where adegrading-enzyme composition prepared by adding collagenase H tothermolysin was used, the liver cells could not be separated. From theseresults, collagenase G was identified as the major protein-degradingenzyme to be used in separating liver cells from a liver tissue.

Reference Example 2: Identification of Substrate for MajorProtein-Degrading Enzyme [Identification of Substrate for Collagenase H]

The pancreas tissue slices (100 mg) of a Lewis rat were treated with 20mM HEPES (pH 8.0) containing a protease inhibitor cocktail (Roche) and 1mM CaCl₂ at 37° C. overnight. Thereafter, the tissue slices were washedwith a buffer and subjected to enzyme degradation with a buffercontaining collagenase H (0.1 mg/mL) for 10 hours at 37° C.

After the enzyme degradation, incubation was performed in 50%acetonitrile containing 100 mM TMPP (Sigma-Aldrich) (10 μL) for 30minutes. Then, acetone precipitation was performed with cold acetone andthereafter centrifugation was performed. The resultant precipitate wasdried and digested with trypsin (10 μL/mg) in a 100 mM ammoniumcarbonate solution, overnight.

After the digest was treated by ZipTip (Millipore), the resultantpeptides were eluted by concentration gradient with 2.5-40% acetonitrilein 10% formic acid and subjected to mass analysis by an LTQ Orbitrap XLmass spectrometer (Terumo Fisher Scientific Inc). Database was searchedby using MASCOT Software 1 program.

Using the TMPP label attached to the N-terminal of the cleaved protein(fragment) as an index, which protein is degraded by collagenase H canbe identified. FIG. 6 shows the TMPP modification ratio of collagen Iand collagen III fragments cleaved with collagenase H. In the figure,data regarding collagen III were plotted within the upper dotted linecircle; whereas data regarding collagen I were plotted within the lowerdotted line circle.

It was revealed that 70% of collagen III and 20% of collagen I aredegraded by collagenase H. By this, it was demonstrated that majorproteins, i.e., substrates for collagenase H, are collagen III andcollagen I.

Example 1: Separation of Cells Depending Upon the Composition of MajorProtein of Biological Tissue [Quantification of Collagen III andCollagen I in Pancreas Tissue and Liver Tissue of Rat]

Tissue slices of a Lewis rat (10 to 13 weeks old) were prepared.Enzymatic immunoassay using labeled antibodies (Chemicon MerckMillipore) against collagen III and collagen I was performed to compareexpression of collagen III and collagen I in the pancreas tissue andliver tissue. As a result, the number of positive staining obtained byimmunohistostaining of collagen III and collagen I in the liver tissuewas significantly lower than that in the pancreas tissue.

[Separation of Cells from Pancreas Tissue and Liver Tissue of Rat]

It was predicted that the major protein of a liver tissue may be aprotein other than collagen III and collagen I, and thus the amount ofcollagenase H in the degrading-enzyme composition for liver cellseparation may be low compared to for pancreatic islet separation.

A liver tissue (10 to 12 g) was treated with a degrading-enzymecomposition (10 mL HBSS) containing protein-degrading enzymes:thermolysin (0.5 mg), recombinant collagenase G (14.4 mg) andcollagenase H (3.6 mg, which is 0.25 times as large as collagenase G) at37° C. for 7 minutes and then subjected to density gradientcentrifugation. Liver cells (5.02±2.11×10⁸) were favorably separated.

A degrading-enzyme composition (10 mL HBSS) containing protein-degradingenzymes: thermolysin (0.3 mg), recombinant collagenase G (8.4 mg) andcollagenase H (2.9 mg, which is 0.35 times as large as collagenase G)was injected through the bile duct and treatment was performed at 37° C.for 14 minutes, and then, density gradient centrifugation was performed.Pancreatic islets (about 4000 islets) were favorably separated.

It was found that, in the separation of pancreatic islets from apancreas tissue where the ratios of collagen III and collagen I inproteins constituting the intercellular matrix are high (the amounts arelarge), a degrading-enzyme composition containing a larger amount ofcollagenase H (0.35 times as large as collagenase G) is preferably used;in contrast, in the separation of liver cells from a liver tissue wherethe contents of collagen III and collagen I are small, adegrading-enzyme composition containing a relatively small amount ofcollagenase H (0.25 times as large as collagenase G) is preferably used.By previously measuring the ratios or amounts of major proteins(collagen III and collagen I) in proteins constituting the intercellularmatrix of a tissue, the amount of protein-degrading enzyme suitably usedfor separating cells from the tissue, more specifically, the amountratio of collagenase H and collagenase G to be used was successfullydetermined.

Example 2: Synergetic effect of NP and CP in separating pancreaticislets from pancreatic islet tissue

A degrading-enzyme composition containing, in addition to collagenase G(8.4 mg) and collagenase H (2.9 mg), any one or two of thermolysin,neutral protease (NP) derived from Clostridium sp. and clostripain (CP)in various amounts to be added, was prepared. Pancreatic islets wereseparated from the pancreatic islet tissue by using the degrading-enzymecomposition prepared in the same manner as in Reference Example 1 andthe numbers of pancreatic islets were compared.

NP and CP were obtained by introducing an NP gene or a CP gene derivedfrom Clostridium histolyticum into Bacillus subtilis and expressing themfollowed by performing purification and then put in use.

In the case where a degrading-enzyme composition containing NP was usedand the case where a degrading-enzyme composition containing thermolysinand CP was used, the number of separated pancreatic islets decreasedcompared to the case where a degrading-enzyme composition containingthermolysin was used. In contrast, in the case where a degrading-enzymecomposition containing NP and CP was used, the number of separatedpancreatic islets significantly increased compared to the case where adegrading-enzyme composition containing thermolysin was used. Inaddition, in the case where a degrading-enzyme composition containing NPand CP was used, the number of separated pancreatic islets was found toincrease depending upon the amounts of NP and CP used.

From these results, it was revealed that neutral protease (NP) derivedfrom Clostridium sp. and protease (CP) having a BAEE degradationactivity synergistically work to exert an effect of increasing thenumber of pancreatic islets to be separated from a pancreatic islettissue.

INDUSTRIAL APPLICABILITY

According to the method for separating cells of the present invention,cells having a high biological activity can be efficiently and stablyseparated from a biological tissue. Accordingly, the method forseparating cells is useful in a wide variety of usages such as celltransplantation, cell strain establishment, therapy, diagnosis andexamination of a disease caused by specific cells.

Furthermore, if specific substrates for individual enzyme componentspresent on a tissue are identified and prepared in the form of a kit,cell separation can be optimized for individual donor tissue in a tailormade manner. In separation of pancreatic islets so far made in the art,a tool having a prefixed composition must be applied to variouspancreatic tissues of donors totally different in matrix composition.However, if highly pure, safe and stable enzyme components are preparedby introduction of biotechnology and specific substrates for the enzymecomponents present on a pancreatic tissue are identified and prepared inthe form of a kit, pancreatic islets that can be adapted to a donor'spancreas can be separated in a tailor made manner.

1. A method for separating liver cells from a liver tissue, using adegrading-enzyme composition, comprising: measuring an abundance ofcollagen I and collagen III in the liver tissue, determining an amountratio of collagenase H to collagenase G derived from Clostridium sp.based on the measured abundance of collagen I and collagen III, whereina higher amount ratio is used when the measured abundance is higher anda lower amount ratio is used when the measured abundance is lower,preparing the degrading-enzyme composition by adding collagenase H andcollagenase G derived from Clostridium sp. with the determined ratio, toa predetermined amount of one or more of a neutral protease or aprotease derived from Clostridium sp., treating the liver tissue withthe degrading-enzyme composition, and separating liver cells from theliver tissue treated with the degrading-enzyme composition, whereincollagenase H degrades collagen I and collagen III, and wherein theamount ratio (H/G) of collagenase H to collagenase G in thedegrading-enzyme composition is 0.25 or less.
 2. The method according toclaim 1, wherein the abundance of collagen I and collagen III in theliver tissue is measured by enzymatic immunoassay using specificantibodies against collagen I and collagen III.
 3. The method accordingto claim 1, wherein the neutral protease is thermolysin or a neutralprotease derived from Clostridium sp.
 4. The method according to claim3, wherein the degrading-enzyme composition contains thermolysin 0.5mg/10 ml, collagenase G 14.4 mg/10 ml and collagenase H 3.6 mg/10 ml.